Journal: Frontiers in Cellular Neuroscience
Article Title: Prevention of noise-induced hearing loss by calpain inhibitor MDL-28170 is associated with upregulation of PI3K/Akt survival signaling pathway
doi: 10.3389/fncel.2023.1199656
Figure Lengend Snippet: MDL-28170 treatment inhibits noise-induced α-fodrin activation in the cochlear tissues. (A) Representative immunoblots from whole cochlear homogenates revealed 240 kDa α-fodrin and the cleaved 150 kDa fragment 3 h after noise exposure. GAPDH (37 kDa) was used as the sample loading control. (B,C) Semi-quantifications of each band density show that the ratio of cleaved to full α-fodrin increased with noise exposure and MDL treatment prevented such an increase (B) , while the cleaved band density divided by GAPDH was not different among the three groups (C) . Data are presented as means + SD; n = 3 in each group with one mouse (two cochleae) per sample, * p < 0.05. (D) Representative images were taken from the basal turn of cochlear epithelia at the time point 3 h after noise exposure. MDL treatment alone showed similar immunolabeling intensity for cleaved α-fodrin (red) in OHCs as the DMSO-treated group. Noise exposure significantly increased cleaved α-fodrin in OHCs, whereas MDL treatment prevented such effects. Phalloidin (green) was a counterstain for visualization of OHCs. The enlarged OHCs allow for better visualization of the immunolabeling. Scale bar = 10 μm. (E) Confocal images used to compare fluorescence intensity by semi-quantification of immunolabeling for cleaved α-fodrin in OHCs were acquired under the same settings and the same processing enhancement for the captured images was used. The person analyzing images was blind to the treatment groups. It confirmed a significant increase after noise exposure, whereas MDL treatment prevented such effects. Data are presented as means + SD; the n is indicated in the bar for each condition with use of one cochlea per mouse. ** p < 0.01, **** p < 0.0001.
Article Snippet: The specimens were first permeabilized in 2% Triton X-100 solution and then blocked with 10% normal goat serum for 30 min each step at room temperature, followed by incubation with primary antibodies: polyclonal rabbit anti-Calpain I (Abcam, Cambridge, UK, #ab39170, 1:50), polyclonal rabbit anti-Calpain II (Abcam #ab39165, 1:50), cleaved α-fodrin (Cell Signaling Technology, Danvers, MA, USA, #2121, 1:50), monoclonal rabbit anti-PI3K, p85α (Millipore, Burlington, MA, USA, #04-403, 1:50), monoclonal rabbit anti-p-Akt (Ser473) (Cell Signaling Technology #4060, 1:50) at 4°C for overnights (24–48 h).
Techniques: Activation Assay, Western Blot, Control, Immunolabeling, Fluorescence
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